Modern pharmacotherapy has been extremely successful in fighting microbial, and especially bacterial, infections, which used to be one of the prime causes of premature death until the middle of the last century. More recently, however, growing concerns over the wide-spread use of highly effective antibiotics have arisen because of the steady increase of bacterial resistance. In fact, over the past 25 years, antibiotic resistance—especially multiple resistance to a broad range of antibiotic compounds—has increased in virtually every species of bacteria examined. It is presently believed that the antibacterial agents of the most advanced type, which are unaffected by common resistance mechanisms, are the compounds which use appears to select for multidrug-resistant mutants.
Based on this development, experts recommend to use antibiotics far more restrictively than in the past, both in agriculture and in human medicine. For instance, minor infections—especially those which are not even typically caused by bacteria, such as the common cold—should not be treated with antibiotics, which should rather be reserved for more serious conditions. Furthermore, it is necessary to develop novel compounds for treating bacterial infections with completely different types of pharmacological activity, preferably with some activity which is independent from bacterial resistance to common antibiotics.
One of the conditions in which the widespread use of antibiotics has been discussed controversially is otitis media, either in its acute form or in its chronic state. It has been shown that the number of patients with otitis media with effusion (OME), i.e. a type of otitis characterized by the presence of fluid in the middle ear without the symptoms of an acute infection, has increased dramatically after the introduction of antibiotic therapy for early acute otitis media (AOM), suggesting that the antibiotics themselves play a part in OME (Lim et al., Laryngoscope 92, 278-286, 1982). It is believed that antibiotics like penicillin interfere with the development of local immune responses, such as with the production of local IgM in the middle ear (Howie et al., Ann. Otol. Rhinol. Laryngol. 85 Suppl. 25, 18-19, 1976). Another disadvantage of antibiotic therapy is that the bacteria are killed, but their toxins are still active.
It has been suggested that, for the treatment of these and other conditions resulting from bacterial or fungal infection, it may be advantageous to use compounds which do not kill the microorganisms or germs themselves, but rather neutralize their toxins and allow the natural host defense mechanisms to control the spread of the infection (Nell, The Role of Endotoxin in the Pathogenesis of Otitis Media With Effusion, PhD Thesis, Leiden, 1999). At the same time, this strategy would support the rapid restoration of impaired mucosal functions.
A major role among microbial toxins, such as fungal toxins and especially bacterial toxins, involved in a number of infectious conditions such as otitis is played by endotoxins, a group of lipopolysaccharides (LPS) found in the cell wall of gram-negative bacteria, consisting of a polysaccharide conjugated with a highly toxic lipid moiety, lipid A. One of the recent therapeutic approaches to treat OME is to administer compounds that neutralize endotoxin, or LPS (Nell, ibid.).
Various compounds capable of neutralizing endotoxin, or LPS, are presently known. For instance, several anti-endotoxin antibodies have been developed, such as HA-1A and E5, a human and a mural monoclonal IgM antibody, respectively. These antibodies have been shown to improve survival rates of patients with some severe conditions such as septic shock (Ziegler et al., New Engl. J. Med. 324, 429-436, 1991). However, their activity and specificity is considered unsatisfactory.
Another group of substances active against endotoxin is derived from a human endogenous protein termed bacterial permeability-increasing protein (BPI), which is stored in the azurophilic granules of neutrophils (Gazzano-Santoro et al., Infection and Immunity 60:11, 4754-4761, 1992). BPI, which is a strongly cationic protein, not only neutralizes free endotoxins, but also inhibits or kills bacteria cells per se by increasing the permeability of their outer membranes. BPI is indeed a potent, natural antibiotic, induced by the presence of LPS and some other triggers including tumor necrosis factor (TNF). However, most of its activity is associated with the immune cells synthesizing it, i.e. polymorphonuclear macrophages.
Several recombinant proteins derived from BPI have also been developed, such as rBPI23 (Kohn et al., 1993) and rBPI21 (Horwitz et al., 1996), which largely represent the N-terminal portions of BPI with molecular weights of 23 and 21 kDa, respectively. The use of BPI and BPI-derived compounds in the treatment of OME has, e.g., been described in WO-A-00/71149.
Another family of natural compounds with antimicrobial activity are the cathelicidins, a class of peptides produced by respiratory epithelial cells, alveolar macrophages, and other tissues. In their native forms, these compounds are linear, α-helical, cystein-free peptides or proteins. Cathelicidins are cationic and comprise a highly conserved signal sequence and pro-region, cathelin. However, their C-terminal domain encoding the mature peptide shows substantial heterogeneity. The peptides may have 12 to 80 amino acids.
The most prominent human cathelicidin is an 18 kDa cationic antimicrobial protein, CAP18. The 37 C-terminal amino acids of CAP18, i.e. peptide LL-37, represent a domain responsible for the high affinity and neutralizing capacity for LPS (Sawa et al., Antimicr. Agents Chemother. 42:12, 3269-3275, 1998). Several truncated peptides derived from CAP18 or LL-37 have been developed and tested, such as those disclosed by Sawa (Sawa et al., ibid.), Gutsmann (Gutsmann et al., Biophys. J. 80, 2935-2945, 2001), and in U.S. Pat. No. 6,040,291 and its European counterpart EP-A-0 955 312.
Further, reference is made to an article of Nagaoka Isao et al. in Clinical and Diagnostic Laboratory Immunology 9 (5) (2002) 972-982 and an article of Kirikae et al. in Infection and Immunity 66 (5) (1998), 1861-1868.
Nagaoka et al. describe the aminoacid sequence of LL-37 and the 18-mer K15-V32 derived therefrom, whereas Kirikae et al. focus on a number of CAP-18 derived patents.
Truncated peptides derived from LL-37 in which the native aminoacid sequence is preserved, and especially such peptides comprising the aminoacid sequence KEFKRIVQRIKDFLRNLV (SEQ ID NO: 1) are hence described in the prior art.
In general, relatively small peptides are preferred over proteins such as CAP18 as lead candidates for therapeutical compounds for several reasons. Firstly, they can more easily be optimized, adapted, and modified to conserve or augment their desired activity and specificity. Secondly, they are easier to obtain or synthesize, and therefore more accessible. Thirdly, they are easier to formulate and deliver, as proteins are often unstable and not bioavailable after non-parenteral administration.